Cell systems having specific interaction of peptide binding pairs

ABSTRACT

This invention relates to modified host cells which express heterologous fused proteins and methods of screening for test samples having peptide binding activity; wherein the modified host cell comprises: (a) a gene sequence encoding a heterologous fusion protein; the fusion protein comprising a first peptide of a peptide binding pair, or segment of the first peptide, which is joined to either a DNA binding domain or its corresponding transcriptional activation domain of a transcriptional activation protein; (b) a gene sequence encoding a heterologous fusion protein, the fusion protein comprising a second peptide of the peptide binding pair in (a), or a segment thereof, fused to either a DNA binding domain or its corresponding transcriptional activation domain, whichever one is not employed in (a); (c) a reporter gene operatively associated with the transcriptional activation protein, or a portion thereof; (d) optionally, a deletion or mutation in the chromosomal DNA of the host cell for the transcriptional activation protein if present in the selected host cell.

This application is a continuation of application Ser. No. 09/305,483,filed May 6, 1999, now U.S. Pat. No. 6,284,519 which is a continuationof application Ser. No. 08/259,609, filed Jun. 14, 1994, issued on Nov.23, 1999, as U.S. Pat. No. 5,989,808, each of which is incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates to novel cells which express heterologous fusedproteins and methods of screening for compounds having peptide-bindingactivity; wherein the methods employ the novel cells of this invention.

BACKGROUND OF THE INVENTION

The specific binding of a pair of peptides to each other triggers a vastnumber of functions in a living cell. For example, the specific bindingof a ligand to a surface receptor serves as the trigger for cellularresponses to many external signals. In mammals, cells respond to a widevariety of circulating peptide hormones, often through singletransmembrane domain receptors. It is certainly recognized that thecytokine receptor superfamily illustrates the diverse aspects ofcellular function, and physiological responses. Recent examinations ofcytokine receptor function have revealed differing ligand-receptorprotein stoichiometries including both 2-protein (ligand/receptor)(Cunningham et al., 1991; Staten et al., 1993) and 3-protein(ligand/receptor/receptor or ligand/receptor/transducer) interactions(Young, 1992; Taga and Kishimoto, 1992; Mui and Miyajima, 1994). Theintricacies of such protein associations have been investigated using invitro, often laborious, methods (Fuh et al., 1992; 1993; Davis et al.,1993) since genetically malleable expression systems have beenunavailable. The present invention is directed to novel modified hostsystems which can be used for such protein investigations, yet the novelsystems are significantly less laborious.

Recently reported systems in the art refer to a “2-hybrid” system asdiscussed by Fields and Song, 1989 and also Chien et al., 1991. The“2-hybrid” system involves differential interactions between theseparable DNA binding and activation domains of the yeasttranscriptional activator, Gal4. Heterologous proteins are expressed ashybrid proteins fused to either half of Gal4 (See FIG. 1; Fields andSong, 1989; Chien et al., 1991 for discussion of the 2-hybrid system).The productive interaction of the heterologous proteins brings the twohalves of the Gal4 protein in close proximity, activating expression ofa scorable reporter gene. To this date, such 2-hybrid systems have beendisclosed as useful for determining whether a first given test peptidesequence has binding activity for the known sequence of a secondpeptide; wherein the affinity of the test peptide for the known peptideis unknown. Studies using such a system have been directed to analyzingintracellular proteins such as transcription factors and kinase-targetprotein interactions (Yang et al., 1992; Durfee et al., 1993; Li et al.,1994).

The novel modified cells of this invention and novel methodsincorporating these cells provide significant advancements for the studyand discovery of peptide mimics, including ligand mimics and receptormimics. At this time no one has developed an efficient and specificscreening system to investigate these areas. By employing in the cell apeptide binding pair for which the binding affinity is known, thepresent invention permits the investigation of peptide binding pairs,such as a ligand and receptor, wherein the peptides bind viaextracellular interactions. The present invention creates exponentialadvantages for the discovery of compounds which can interact as ligandsfor specific receptors or transducers. Potential ligands include, butare not limited to, mammalian hormones with the receptors being acognate extracellular ligand-binding peptide. Furthermore, the presentinvention describes the use of cell systems which express multipleheterologous proteins, including the two heterologous fused proteins toestablish the specific and reversible binding of the ligand andreceptor. The specific interaction of the above-described binding isreadily detected by a measurable change in cellular phenotype, e.g.growth on selective medium.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to novel modified host cellsfor the expression of heterologous fusion proteins. The novel modifiedhost cells comprise:

a) a gene sequence encoding a heterologous fusion protein; said fusionprotein comprising a first peptide of a peptide binding pair, or segmentof said first peptide, which is joined to either a DNA binding domain orits corresponding transcriptional activation domain of a transcriptionalactivation protein;

b) a gene sequence encoding a heterologous fusion protein, said fusionprotein comprising a second peptide of the peptide binding pair in (a),or a segment of said second peptide, fused to either a DNA bindingdomain or its corresponding transcriptional activation domain, whicheverone is not employed in (a);

c) a reporter gene operatively associated with the transcriptionalactivation protein, or a portion thereof.

d) optionally, a deletion or mutation in the chromosomal DNA of theyeast host cell for the transcriptional activation protein if present inthe host cell.

These novel modified host cells of the present invention can be used todetermine the interaction of a test sample with a selected peptide of apeptide binding pair; e.g. the cell can be used to determine theinteraction of a test sample with selected ligand or receptor.

A second aspect of the present invention relates to novel modified cellsand screening methods which indicate the interaction of a test samplewith a selected peptide and receptor by a recognizable change inphenotype. The cell exhibits the change in phenotype only in thepresence of test compound having binding affinity for a peptide of thepeptide binding pair, e.g. binding affinity for a ligand or itsreceptor.

A third aspect of the present invention relates to novel cells andscreening methods which permit determining to which peptide of a peptidebinding pair a test sample binds.

A fourth aspect of the present invention relates to novel cells whichexpress three or more heterologous components for the study of higherorder multi-protein associations between three or more peptides (e.g.such as the study of ligand dependent dimerization).

Defined Terms:

The term peptide binding pair refers to any pair of peptides having aknown binding affinity for which the DNA sequence is known or can bededuced. The peptides of the peptide binding pair must exhibitpreferential binding for each other over any other components of themodified cell.

The term peptide as used in the above summary and herein means anypeptide, polypeptide or protein, unless stated otherwise. As notedabove, the peptides of a peptide binding pair can be a ligand and itscorresponding receptor, or a ligand and any peptide having a knownbinding affinity for the ligand.

Heterologous as used in the above summary and herein means peptideswhich (1) are not expressed by the naturally-occurring host cell or (2)are expressed by the modified host cell by an expression method otherthan the expression method by which the host cell would normally expressthe peptide.

Unless specified otherwise, the term receptor as used herein encompassesthe terms receptor, soluble receptor, transducer and binding protein. Ina preferred embodiments of the invention, the receptor employed is areceptor, or soluble receptor, with receptor being more preferred.

Receptor, as used herein means plasma membrane proteins that bindspecific molecules, such as growth factors, hormones orneurotransmitters, and then transmits a signal to the cells' interiorthat causes a cell to respond in a specific manner. This includes singletransmembrane proteins.

Soluble receptor means a non-transmembrane form of a receptor which isable to bind ligand. These are receptors released from a cell either byproteolysis or by alternatively spliced mRNA.

Binding protein means proteins that demonstrate binding affinity for aspecific ligand. Binding proteins may be produced from separate anddistinct genes. For a given ligand, the binding proteins that areproduced from specific genes are distinct from the ligand binding domainof the receptor, or its soluble receptor.

Transducer means a molecule that allows the conversion of one kind ofsignal into another, and the molecule is readily known as a transducerfor one or more the peptides of a peptide binding pair, e.g. atransducer for a ligand/receptor group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a cell which expresses from separateplasmids two heterologous fused proteins (one being the ligand fused tothe activation domain of a transcriptional activation protein and theother fused protein being a receptor fused to the DNA binding domain ofthe transcriptional activation protein). The Figure shows the expressionof the two fused proteins and the binding of the ligand and receptor,which brings together the binding domain and activation domain,reconstituting the transcriptional activation protein. Once thetranscriptional activation protein is reconstituted and anchored by theDNA binding domain to the Upstream Activation Sequences (UAS) site,transcription of the reporter gene (HIS3) is initiated.

FIG. 2 contains photographs of plates which show the results of thegrowth experiments conducted in example 1 for the stains CY722, CY723,CY724, and CY781 on non-selective medium and selective medium,photographs A and B, respectively.

FIG. 3 is a schematic diagram of a dimer model, in which the ligandbinds to a dual receptor system. The schematic diagram depicts a cellwhich expresses proteins from three separate plasmids. Two heterologousfused proteins (one fused protein being a first receptor fused to theactivation domain of a transcriptional activation protein and the otherfused protein being a second receptor fused to the DNA binding domain ofthe transcriptional activation protein) are expressed and free ligand(i.e. ligand which is not fused to either of the two domains of thetranscriptional activation protein) is expressed from a third plasmid.The figure shows the expression of the two fused proteins and thebinding of the free ligand and two receptor fusions which bringstogether the binding domain and activation domain, reconstituting thetranscriptional activation protein. Once the transcriptional activationprotein is reconstituted and anchored by the DNA binding domain to theUpstream Activation Sequences (UAS) site, transcription of the receptorgene (HIS3) is initiated.

FIG. 4 is a photograph of the growth plate obtained for the strainsCY846 and CY847 from Example 3, showing ligand-dependent stimulation ofreceptor dimerization.

DETAILED DESCRIPTION OF THE INVENTION

The modified cell of this invention employs a host cell. An effectivehost cell for use in the present invention simply requires that it isdefined genetically in order to engineer the appropriate expression ofheterologous fused proteins, reporter(s) and any other desired geneticmanipulations. The host cell can be any eukaryotic cell, vertebrate ornon-vertebrate. The host cell can be mammalian as well as amphibian,e.g. a Xenopus egg cell. Preferably, the host cell is a fungal cell,e.g. Aspergillus or Neurospora. In more preferred embodiments the hostcell is a yeast cell. In alternatively preferred embodiments the yeasthost cell is Saccharomyces cerivisiae, Schizosaccharomyces pombe orPichia pastoris.

The modified host cell employs at least two genes for expressingseparately the two heterologous fusion proteins. One of these fusionproteins comprises a first peptide of a peptide binding pair, or segmentof said first peptide, which is joined to either a DNA binding domain,or its corresponding transcriptional activation domain, of atranscriptional activation protein. A second fusion protein comprisingthe second peptide of the peptide binding pair, or a segment thereof.The second peptide is fused to either a DNA binding domain or itscorresponding transcriptional activation domain, whichever one is notemployed in the first heterologous fused protein. The activity of thebinding between the peptides of the peptide binding pairs is monitoredby the use of a reporter gene, which is operatively associated with thetranscriptional activation protein employed in the two fusion proteins.

The transcriptional activation protein can vary widely as long as theDNA binding domains and the activation domains are known or can bededuced by available scientific methods. The transcriptional activationprotein can be any protein having two components, a DNA bindingcomponent and an activation component, wherein the transcriptionalactivation protein contains an acidic alpha helix for the activation oftranscription. Preferably, the transcriptional activation protein isselected from Gal4, Gen4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1,Ppr1, Arg81, Lac9, Qa1F, VP16, LexA, non-mammalian nuclear receptors(e.g. ecdysone) or mammalian nuclear receptors (e.g. estrogen,androgens, glucocorticoids, minearalocorticoids, retinoic acid andprogesterone; see also Picard et al., 1990). Preferably, thetranscriptional activation protein is a yeast protein, and morepreferably, the transcriptional yeast protein is selected from Gal4,Gcn4 or Adr1. It is noted that any DNA binding protein can be used whichfunctions with an activation domain. A DNA binding protein can besubstituted for the DNA binding domain of a transcriptional activationprotein if the recognition sequences operatively associated with thereporter gene are correspondingly engineered. Illustrative of non-yeastDNA binding proteins are mammalian steroid receptors and bacterial LexA(see Wilson et al., 1990)

The reporter gene is generally selected in order that the binding of thedomains of the transcriptional activation protein can be monitored bywell-known and straightforward techniques. Preferably, the reporter geneis selected based on its cost, ease of measuring its activity and lowbackground (i.e. the activity can be determined at relatively low levelsof expression of the reporter gene because of a high signal tobackground ratio and/or a relatively low or no un-induced activity). Thereporter can be any reporter for which its activity can be detected byany means available. Illustrative of reporters which can be used in thepresent invention are reporter genes selected from the group of:

a) lacZ, Luciferase gene, green fluorescent protein gene, CATchloramphenicol acetyltransferase

b) genes complementing auxotrophies, such as HIS, URA, LEU, ARG, MET,ADE, LYS, TRP.

c) gene conferring antibiotic resistance, such as neo^(r), KAN,

d) genes conferring sensitivity to a chemical such as CYH2(cycloheximide sensitivity, CAN1 (canovanine sensitivity). In manyembodiments it may be convenient for the reporter gene to prevent growth(CYH2). Preferably, the activity of the reporter gene is indicated bycolorimetric or fluorescent methods and/or by measuring growth of theyeast cell.

As noted previously, the peptide employed in the modified cell is apeptide of a peptide binding pair for which the DNA sequence is known aswell as the sequence of the second peptide of the binding pair. Thepeptides can also be peptides of a peptide binding complex whichcontains two or more peptides which bind each other to form the bindingcomplex. The peptides of the peptide binding pair can be a specificligand and a corresponding receptor or any other peptides which bind toeach other preferentially, such subunits of an enzyme.

One of the significant advantages of this invention is the discoverythat the modified cell employing the DNA binding and activation domainsof a transcriptional protein can be used to monitor the binding ofpeptides of a peptide binding pair which bind through extracellularinteraction. Certainly, if desired peptides which bind throughintracellular interactions can also be employed in any of the novelmodified cells and methods of this intention. The peptide can be from amammalian cell or non-mammalian cell. One of the most importantembodiments of the present invention relates to the application of thenovel modified cells and corresponding screening methods of thisinvention for studying numerous mammalian peptide interactions. Themammalian peptides include mammalian ligand/receptor interactions, suchas hormone/receptor interactions. Illustrative of peptide hormones whichcan be used in the present invention are peptides selected from, but notlimited to, one of the following groups: (a) the group consisting ofcytokines, interleukines, hematopoietic growth factors, insulin,insulin-like growth factors, growth hormone, prolactin, interferons, andgrowth factors; (b) ligands for G-protein coupled receptors; (c) ligandsfor nonvertebrate receptors; (d) ligands for guanylyl cyclase receptors;and (e) ligands for tyrosine phosphatase receptors.

In alternative embodiments, the peptide is a growth factor selected fromepidermal GF, nerve GF, leukemia inhibitory factor, fibroblast GF,platelet-derived GF, vascular endothelial GF, tumor necrosis factor,oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor,placental lactogen and transforming GFβ.

In various preferred embodiments the peptide hormone is a ligand for aG-protein coupled receptor, such as growth hormone releasing factor,secretin, vasoactive inhibitory peptide, glucagon, thyrotropin,interleukin-8, luteinizing hormone (LH) and follicle stimulating hormone(FSH).

In additional alternative embodiments the peptide employed is anonvertebrate peptide, such as those selected from the group consistingof plant systemin and insect differentiation peptides. However, inpreferred embodiments the peptide is selected from the group consistingof mammalian peptides, and more preferably, mammalian peptide hormones.

It is also noted that specific types of receptors may also be a peptideof a peptide binding pair or peptide binding complex. Illustrative ofvarious receptors are those selected from one of the following groups:(a) a cell adhesion molecule; (b) an immunomodulatory, antigenrecognition or presentation molecule or other related peptides.Illustrative of cell adhesion molecules are ICAM, VCAM, ECAM,fibronectin, integrin, selectin and fibrinogen. Illustrative of animmunomodulatory, antigen recognition or presentation molecule are Tcell receptor complex, B cell receptor complex, Fc receptors, majorhistocompatibility complex I, major histocompatibility complex II, CD4,CD8, CD27, CD30, MAC complex.

It is also noted that specific types of transducers may also be used asa peptide of a peptide binding pair or peptide binding complex. Thetransducer proteins employed can be any transducer protein which bindsat least one of the peptides of the peptide binding pair or peptidebinding complex. Transducer proteins include gp130, kh97, AIC2A, AIC2B.

Preferably, the heterologous fused proteins are expressed bytransformation of the yeast cell with an autonomously-replicatingplasmid capable of expressing the fusion protein although they can beexpressed by chromosomal modification.

As noted, the screening methods of this invention are designed in orderto detect the ability of a test sample to affect the binding of apeptide binding pair, e.g. ligand-receptor interaction. Basically, themethod comprises determining the activity of the reporter gene uponadding a test sample to a modified host cell of the present inventionunder conditions suitable to detect the activity in the presence of asample or under a condition for which the modified host cell exhibitssuch activity only in the presence of a sample having bindinginteraction with the peptide binding pair. Preferably, the activity ofthe reporter gene is determined by measuring a change in selectedphenotype which is directly correlated to activity of the reporter.

The novel modified cells of this invention are readily applied invarious screening methods for determining the binding ability of a testsample. The test sample may be a peptide, which is preferably about twoamino acids in length, or a non-peptide chemical compound. Thenon-peptide test sample includes compounds, complexes and salts as wellas natural product samples, such as plant extracts and materialsobtained from fermentation broths. The modified host cells are culturedunder suitable conditions for growth to study the interaction of a testsample on the binding interaction of the peptide binding pair. Themodified host cells are placed in a growth medium, which preferablycontains agar, with the test sample applied to the surface of the growthmedium. The growth medium is preferably a conventional liquid medium ofgrowth reagents and water, such as yeast synthetic medium (YSM availablefrom BIO101 (also see Rose et al., Methods in Yeast Genetics, 1990).

One of the embodiments of the present invention is directed to a novelmodified host cell and screening method which indicate the interactionof a test compound with a selected peptide binding pair by arecognizable change in phenotype. This modified host cell exhibits thechange in phenotype only in the presence of test compound having bindingaffinity for one of the peptides of the peptide binding pair. This hostcell is referred to herein as a “rescue” system. Normally, a cellresponse is exhibited when the two domains of the transcriptionalactivation protein interact. However, in a rescue system a positiveindication of change in the phenotype does not occur when the twodomains of the transcriptional activation protein interact. A positiveindication of change in the phenotype occurs only when a test sampleinterrupts the interaction of the two domains of the transcriptionalactivation protein. In a rescue system, a modified host cell is capableof expressing at least two heterologous fusion proteins. Further, thehost cell comprises: a reporter gene operatively associated with thetranscriptional activation protein; wherein said reporter gene preventsthe exhibition of a specific phenotype on a selective medium due to theexpression of the transcriptional activation protein, or a portionthereof. A mutation in the chromosomal DNA of the host cell allows forreversal of the detectable phenotype, on the selective medium, in theabsence of expression of the reporter gene. If needed, there is adeletion or mutation in the chromosomal DNA of the host cell for atranscriptional activation protein in order that transcriptionalactivation only occurs upon productive interaction of the selectedbinding pair. Only when a test sample interrupts the interaction of thetwo-domains of the transcriptional activation protein will the modifiedcell grow or survive, or exhibit another selected phenotype. Preferably,the phenotype corresponds to the growth of the cell.

Once a screening method, as discussed above, is used to determinewhether a test sample interacts with, or rather disrupts, the peptidebinding observed in the absence of a test sample, a secondary screen isemployed to determine the specific binding affinity of the test sample,i.e. to which peptide of the peptide binding pair the test sample binds.The secondary screens employ the novel cells of this invention whereincells are adapted to exhibit a phenotype, or phenotypic change only inthe presence of a test sample which binds one peptide of the peptidebinding pair. One of the preferred methods for determining the specificbinding characteristics of the test sample involves employing cellswhich contain an effective (relatively high) copy number of eitherfusion protein containing one of the peptides. An effective copy numberis any copy number sufficient to enable determination of the specificbinding of the test sample. Preferably, the gene copy number is at leastabout 5, and preferably ranges from about 5 to about 50, with the highercopy numbers being the most preferred. The other fusion protein ismaintained at a relatively low (1 to about 2 copies per cell) by eitherintegration into a cell chromosome or by utilizing chromosomalcentromeric sequences on the expression plasmid. If a high copy numberof a first peptide is used in a cell of this invention, the cell will bemore sensitive to the presence of the test sample which binds the secondpeptide of the peptide binding pair since the limiting amount of secondpeptide determines the level of the activity of the reporter gene (i.e.the change in phenotype observed). Conversely, if a high copy number ofa gene encoding the second peptide of a peptide binding pair is used,the cell will be more sensitive to the presence of a test sample whichbinds the first peptide since the limiting amount of the second peptidedetermines the level of the activity of the reporter gene (i.e. thechange in phenotype observed). A direct comparison of effects of a testcompound on the phenotypes of the two strains (receptor>>>ligand versusligand>>>receptor) demonstrates the specific protein interaction of thecompound. As discussed supra, the genes expressing the peptides as wellas the reporter gene are preferably expressed by transformation of theyeast cell with an autonomously-replicating plasmid.

An additional modified host cell of this invention is directed to cellswhich can be used to study peptides ligands which employ dual receptorsor a receptor and a transducer for activation or transmission of asignal from the binding of multiple peptide binding components, i.e.three or more peptide binding components.

Receptor dimerization is a critical first step for signal transductionfor certain classes of receptors. Dimer receptor structures can becomposed of identical receptor units (examples: insulin receptor, IGF-Ireceptor, PDGF receptor, kinase inert domain receptor (KDR), or colonystimulating factor (CSF)-I receptor) or non-identical receptor units(example: IL-6R+gp130; insulin-IGF-I hybrid receptor; LIF+gp130;CNTF+gp130; various interferon receptors).

The components of a modified host cell for monitoring the bindingactivity of a peptide having “dual receptor” system are as follows: thegene sequence (a) is a gene sequence encoding a heterologous fusionprotein; said fusion protein comprising one peptide of a multiplepeptide binding complex, or segment of said peptide, which is joined toeither a DNA binding domain or its corresponding transcriptionalactivation domain of a transcriptional activation protein; and the genesequence (b) is a gene sequence encoding a heterologous fusion protein;said fusion protein comprising a second peptide of said multiple peptidebinding complex, or a segment of said receptor, fused to either a DNAbinding domain or its corresponding transcriptional activation domain,whichever one is not employed in (a). The modified host cell forstudying a multiple peptide binding complex, such as a dual receptorsystem, also comprises an appropriate reporter gene and chromosomalmutations for specific analysis of the peptide (ligand/receptor)interaction as discussed infra. One can express a third peptide (e.g. aligand) to establish a control for comparative or competitive testing.

As noted above, for the study of multiple binding peptide complexes,i.e. higher-order proteins which contain three or more peptides, one canactually use the modified host cell of the present invention to expressthree or more peptides. In the case of a tripeptide binding complex, anytwo of the peptides can be fused to the two components of thetranscriptional activation protein. For example, to study theinteraction of a ligand which interacts via receptor dimerization, onecan express the receptors as fused proteins with the ligand beingexpressed as a nonfusion protein. This host cell system can be also beapplied in studying multiprotein enzyme complexes. For any multiplepeptide binding complex, one can identify novel peptides which interactwith the complex by expressing novel proteins from random complementaryDNA sequences (e.g. a cDNA library) fused to one of the domains of atranscriptional activation protein. In such a system, one of the knownpeptides of the peptide binding complex is fused to the other domain ofthe transcriptional activation protein while other units of the peptidebinding complex are expressed as nonfusion peptides. It is further notedthat the number of peptides expressed by the modified host cell shouldonly be limited by the available detection means and the capacity of thehost cell.

The novel screening methods can be utilized to identify compoundsinteracting with any peptide binding pair, e.g. any receptor and/orligand. Also, this modified cell system with a reporter gene to create ascreen can be applied to any protein-protein interaction to discovernovel compounds that disrupt that interaction. As specific examples: a)protein kinases implicated in cancers can be inserted into the system torapidly screen for novel compounds that block the kinase-targetinteraction and thus may serve as unique cancer therapeutics; b) viralcoat proteins, such as human immunodeficiency virus glycoproteins, andcorresponding cell surface receptor proteins, such as CD4, can beinserted into the system to rapidly screen for compounds that disruptthis interaction, and may serve as anti-viral agents; c) the twosubunits for the Plasmodium ribonucleotide reductase enzyme can beexpressed in the system to screen for compounds which prevent thisspecific protein association and thus may serve as novel anti-malarialagents.

The following examples are provided to further illustrate variousaspects of the present invention. They are not to be construed aslimiting the invention.

EXAMPLE 1 Specific and Reversible Ligand—Receptor Interaction

Genes encoding fusion proteins are generated by cloning growth hormone(GH) and growth hormone receptor (GHR) cDNA sequences into plasmidscontaining the coding region for the domains Gal4. DNA binding domain(Gal4) fusions are constructed in pAS2, which is described in WadeHarper et al. Gene activation domain (Gal4) fusions are constructed inpACT-II, which is identical to pACT (described in Durfee et al., 1993)except with a modification of the polylinker region. Into the Bgl IIsite is added the following sequence: Bgl II-Hemagglutininepitope-NdeI-NcoI-SmaI-BamHI-EcoRI-XhoI-Bgl II, as adapted from thepolylinker sequence of pAS2 (Wade Harper et al., 1993). The cDNAencoding the mature peptide for porcine GH is generated using standardpolymerase chain reaction (PCR) techniques (see Finney, 1993).

Oligonucleotides prepared on an ABI oligosynthesizer are designedaccording to the published cDNA sequence for pig GH (see Su andEl-Gewely, 1998). A 30 base 5′ oligonucleotide contains a NcoI site(5′-CATGCCATGGAGGCCTTCCCAGCCATGCCC 3′ SEQ ID NO: 1) and a 27 base 3′oligonucleotide contains a BamHI site (5′-CGGGATCCGCAACTAGAAGGCACAGCT-3′SEQ ID NO: 2). The GH cDNA is generated using a pig pituitary lambdagt11 library as template source. A 540 bp fragment is obtained, ligatedinto pCR II vector (Invitrogen Corp.), recombinants are confirmed byrestriction enzyme digest, and the DNA produced as described in Maniatuset al., 1982. The cDNA sequence is confirmed by dye-deoxy terminatorreaction using reagents and protocols from Perkin-Elmer Cetus Corp. andan ABI 373A automated sequencers. The GH cDNA is directionally clonedinto pACT-II via NcoI and BamHI sites. The cDNA encoding theextracellular domain of the GHR is generated using standard PCR methods.A 33 base-5′ oligonucleotide containing a NcoI site(5′-CATGCCATGGAGATGTTTCCTGGAAGTGGGGCT-3′ SEQ ID NO: 3) and a 39 base 3′oligonucleotide containing a termination codon, followed by a NcoI site(5′-CATGCCATGGCCTACCGGAAATCTTCTTCACATGCTGCC-3′ SEQ ID NO: 4) are used togenerate a 742 bp fragment encoding amino acids 1-247 of the rat GHR(Baumbach et al., 1989). This GHR cDNA is cloned into pCRII vector aspreviously described above, and then subcloned into the NcoI site of thepAS2 vector. DNA of the final recombinant vectors is transformed intoyeast strain(s) by the lithium acetate method (Rose et al., 1980).

A yeast host (Y190) containing a UAS_(GAL)-HIS reporter gene is preparedaccording to the procedure described in Wade Harper et al., 1993. Thegenotype of strain Y190 is MATa leu2-3, 112 ura3-52 trp1-901 his3d200ade2-101 gal4 gal80 URA3::GAL-lacZ LYS2::GAL-HIS3 cyh^(y). Strain Y190is transformed with both fusion constructs or with a single fusionconstruct plus the opposing vector containing no heterologous sequences.All strains are found to exhibit equal growth on nonselective medium(FIG. 2A). These strains are then tested for growth on selective medium(i.e. a growth medium lacking an amino acid which is synthesized byactivation of the reporter gene). Only the strain containing both hybridproteins (CY722) is able to grow while the strains containing either theligand or receptor fusion alone do not grow (CY724 and CY723,respectively; FIG. 2B). Two independent samples of each strain arestreaked on synthetic medium containing 2% glucose, yeast nitrogen base,ammonium sulfate, 0.1 mM adenine and 60 mM 3-amino-triazole (plate B) oron the same medium supplemented with histidine (plate A). Plate A isincubated at 30 C for three days; plate B for five days. These resultsdemonstrate that GH and GHR can mediate the Gal4-dependent activation ofthe reporter gene in an interaction suggestive of ligand-receptorbinding.

EXAMPLE 1A Competing Expressed Free Ligand (GH) in the Presence of GHand GHR Fusion Proteins

To substantiate the apparent binding of GH to its receptor in theforeign environment of a yeast nucleus, the system is modified to add athird plasmid mediating expression of “free” ligand to show that the GHpeptide competes with the GH-Gal4 fusion protein, reversing the 1-hybridinteraction shown in Example 1. The parental strain Y190 (Wade Harper etal., 1993) is grown on a medium containing 5-fluoro-orotic acid toselect for derivatives that spontaneously lose the URA3 gene (see Roseet al., 1990). The resultant strain, designated CY770, is utilized forall experiments examining effects of protein expressed concurrently fromthe third component (i.e. third plasmid). The cDNA encoding GH isgenerated by PCR methods using a 38 base 5′ oligonucleotide containingan EcoRI site (5′-CCGAATTCAAAATGGCCTTCCCAGCCATGCCCTTGTCC-3′ SEQ ID NO:5) and a 26 base 3′ oligonucleotide containing a HindIII site (5′CCAAGCTTCAACTAGAAGGCACAGCT-3′ SEQ ID NO: 6) for subsequent subcloninginto the vector pCUP. pCUP is an inducible yeast expression vectorderived from pRS316 (Hill et al., 1996). Briefly, this vector isconstructed by inserting the 3′ end of the yeast PGK gene (from pPGK;Kang et al., 1990) into the pRS316 cloning region as a BamHI-SalIfragment to serve as a transcriptional terminator. To this plasmid, theCUP1 promoter region (Butt et al., 1984) is amplified by PCR as aSacI-EcoRI fragment and inserted into corresponding sites of the plasmidto create pCUP. The GH expression plasmid (GH-pCUP) is thenco-transformed with the GH and GHR fusion constructs into strain CY770to generate CY781. Concurrent expression of free GH with the GH and GHRfusion proteins (CY781) is shown to block GH-GHR-dependent cell growthon selective medium (FIG. 2B). This experiment typifies an in vivocompetition assay and demonstrates the reversibility of the observedligand-receptor interaction.

EXAMPLE 1B Binding of Peptide Hormone Prolactin (PRL) and its Receptor

To expand and validate this technology, a similar system was developedusing the peptide hormone prolactin (PRL) and its receptor. Prolactin isstructurally related to GH and the prolactin receptor (PRLR) is also amember of the cytokine receptor superfamily. Unlike human GH,sub-primate GH does not readily bind the PRLR (Young and Bazer, 1989);nor does PRL readily bind the GHR (Leung et al., 1987). Mature porcinePRL is generated as a fusion to the GAL4 activation domain.Oligonucleotides are designed to pig PRL (obtained from Genbank X14068),and used to generate the mature pig PRL protein hormone from a pigpituitary lambda gt11 library using standard PCR methods. A 31 base 5′oligonucleotide includes an EcoRI site(5′-CGGAATTCTGCCCATCTGCCCCAGCGGGCCT-3′ SEQ ID NO: 7) and corresponds tosequences encoding amino acids 1-7. A 30 base 3′ oligonucleotidecontains an EcoRI site (5′-GAATTCACGTGGGCTTAGCAGTTGCTGTCG-3′ SEQ ID NO:8) and corresponds to a region of cDNA 3′ to the endogenous terminationcodon. A 600 bp fragment is obtained, ligated into pCR II vector, andconfirmed by restriction enzyme digest and sequence analysis. The PRLcDNA is cloned into pACT-II via the EcoRI site.

The extracellular domain of the porcine PRL receptor (PRLR) is generatedas a fusion to GAL4 DNA binding domain. Oligonucleotides are designedbased on sequence of the mouse PRLR (Davis and Linzer, 1989) A 31 base5′ oligonucleotide contains a SmaI site(5′-TCCCCCGGGGATGTCATCTGCACTTGCTTAC-3′ SEQ ID NO: 9) while the 31 base3′ oligonucleotide contains a termination codon followed by a SalI site(5′ TCCGTCGACGGTCTTTCAAGGTGAAGTCATT-3′ SEQ ID NO: 10). Theseoligonucleotides flank the extracellular domain of the PRLR, encodingamino acids 1-229. A pig pituitary lambda gt11 library is used astemplate source. Using standard PCR methods, a 687 bp fragment isgenerated, ligated into pCRII, and the nucleotide sequence is confirmed.The PRLR cDNA is cloned into the pAS2 vector via the SmaI and SalIrestriction sites.

Strain Y190 was transformed with the PRL or PRLR fusion expressionplasmids either alone (CY727 or CY728, respectively) or together(CY726). Cells expressing both the PRL and PRLR fusions are able to growon selective medium while the strains containing either the ligand orreceptor fusion alone can not. These results mirror those observed inthe GH-GHR system in the examples above and establish the generalutility of the 2-hybrid system for examination of ligand binding tomembers of this receptor superfamily.

EXAMPLE 1C Additional Confirmation of Ligand-Receptor Specificity forthe Novel Yeast Host Cell System

Additional strains are developed to assess ligand-receptor specificity.URA minus strains expressing GH and GHR fusion proteins are transformedwith pCUP or PRL-pCUP; while strains expressing PRL and PRLR fusionproteins are transformed with pCUP, or PRL-pCUP. Briefly, PRL-pCUP isconstructed in a fashion similar to that described for GH-pCUP. The PRLcDNA is generated by PCR using a 33 base 5′ oligonucleotide with anEcoRI site (5′-GAATTCAAAATGCTGCCCATCTGCCCCAGCGGG-3′ SEQ ID NO: 11) andthe 3′ oligonucleotide in example 1B. The resulting fragment isintroduced into pCUP via the EcoRI site. As demonstrated in the aboveExamples, a strain expressing the GH and GHR fusions with no competitorgrows on selective medium and this growth is abolished with coexpressionof free GH. The prolactin experiment produces similar results whichconfirm the specificity of the ligand-receptor binding in the yeastcell. A strain carrying PRL and PRLR fusions (CY787) can grow onselective medium and this growth is abrogated by expression of free PRL(CY786; Table 1).

To test selectivity of the GHR, a strain containing the GH and GHRfusions is transformed with PRL-pCUP. This strain grows on selectivemedium (CY785; Table 1). These data indicate that GH binding to itsreceptor in this system can be efficiently competed by excess GH (CY751)binding but not by the related PRL peptide (CY755). The results from theabove experiments, expressing three heterologous proteins, illustratesthe specificity of ligand-receptor interaction(s) in the system of thisinvention.

TABLE 1 Strain list and bioassay results Designation AD fusion BD fusionpCUP Growth CY700 — — — 0 CY722 GH GHR — + CY723 vector GHR — 0 CY724 GHvector — 0 CY726 PRL PRLR — + CY770 — — — 0 CY781 GH GHR GH 0 CY784 GHGHR vector + CY785 GH GHR PRL + CY786 PRL PRLR PRL 0 CY787 PRL PRLRvector + All yeast strains are derived from strain Y190 (Wade Harper etal. 1993). The genotype is MATa gal4 gcl80 his3 trp1-901 ade2-101ura3-52 leu2-3.112 URA3::GAL-lacZ LYS2::GAL-HIS3 cyh. Strains withnumber designations equal to or greater than 770 do not have theURA3::GAL-lacZ gene. A dash indicates that a strain does not contain thedenoted plasmid. AD fusions are pACT derivatives; GH or PRL fused to theGa14 activation domain. BD fusions are pAS2 derivatives; extracellulardomains of GH or PRL receptors fused to the DNA binding domain of Ga14.pCUP denotes peptides expressed from the pCUP plasmid. Summary ofbioassay results. Each strain is grown on selective medium for 3 to 5days at 30 C. then scored for cell growth, indicated by a plus.

EXAMPLE 2 Screen for Compounds Disrupting Ligand-Receptor Interaction

Low-copy-number plasmids expressing GHR- or GH-Gal4 fusion proteins(pOZ153 and pOZ152, respectively) are constructed to reduce expressionof these proteins. In addition, a novel reporter gene is constructedthat prevents cell proliferation on selective medium unless expressionis abrogated. To construct the GHR fusion expression plasmid, aSacI-BamHI restriction fragment containing a yeast constitutive promoterand GAL4 sequences is isolated from pAS1 (Durfee et al., 1993) andcloned into pUN30 (Elledge and Davis, 1988). The extracellular domain ofGHR is then fused to GAL4 by ligation as an NcoI fragment as describedin Example 1 to create pOZ153. To construct the GH fusion expressionconstruct the entire GH-Gal4 region with promoter and terminatorsequences is isolated from the plasmid described in Example 1 as aPvuI-SalI fragment. This DNA segment is cloned into pUN100 (Elledge andDavis, 1988) generating pOZ152. A reporter gene is constructed byisolating the yeast CYH2 coding region and operatively linking it to aGAL promoter in a yeast expression plasmid. Briefly, the GAL1 promoterregion is inserted into YEp352 (Hill et al., 1986) as a 685 bpEcoRI-BamHI fragment. CYH2 sequences are amplified by PCR usingoligonucleotides primers (5′-GGATCCAATCAAGAATGCCTTCCAGAT-3′ SEQ ID NO:12 and 5′-GCATGCGTCATAGAAATAATACAG-3′ SEQ ID NO: 13) and pAS2 as thetemplate. The PCR product is digested with BamHI plus SphI and clonedinto the corresponding sites in the YEp352-GAL vector. These plasmidsare transformed into yeast strain CY770 which carries a mutation at thechromosomal cyh2 gene rendering the strain resistant to the proteinsynthesis inhibitor cycloheximide. The presence of all three plasmids isnecessary to confer cycloheximide sensitivity (cyh5).

The strain (CY857) containing the ligand and receptor fusion plasmidsplus the reporter plasmid forms the basis of a simple primary screen forcompounds that disrupt the binding of GH to its receptor. Strain CY857is embedded in standard yeast growth medium containing 10.0 μg/mlcycloheximide. Due to the ligand/receptor interaction driving expressionof the CYH2 reporter gene, the strain is cyh· and thus unable to grow.Chemical compounds are placed on this test medium. Compounds whichimpair GH-GHR binding are identified by the growth of cells surroundingthe compounds because in the absence of CYH2 expression the cells becomeresistant to cycloheximide present in the medium.

Secondary Screen to Determine Target of Sample

Disruption of ligand-receptor binding in this assay can result fromreaction of the compound with either the receptor or ligand fusioncomponent. The specific target of the novel compound is determined by asimple secondary assay utilizing strains over-expressing one of thefusion proteins. Strain CY858 expresses the GHR-GAL4 fusion in largeexcess due to the construct being maintained within the cells at highcopy number (pOZ149), while the GH-fusion (pOZ152) is maintained atlevels similar to the base strain (CY857). Conversely, strain CY859expresses the GH-GAL4 fusion in large excess due to this construct beingmaintained within the cells at high copy number (pKY14), while the GHRfusion (pOZ153) is maintained at levels similar to base strain (CY857).Compounds rescuing growth in the primary screen using CY857 (GH and GHRfusion expressed on low copy numbers plasmids) are then assayed in thesame manner using CY858 (GHR>>GH) or CY859 (GH>>GHR) as the test strain.For example, when ligand-receptor binding is inhibited by a compoundreacting with the GHR, the secondary screen will demonstrate adetectable change for the phenotype measured. Secondary testing of therescuing compound on strain CY858 which overexpresses the GHR fusionproduces a smaller growth in the presence of the compound than thatobserved for CY859. This detectable change in the measured phenotypeoccurs because the overabundance of GHR titrates the compound therebyincreasing CYH2 expression and inhibiting cell growth. CY859 produces adetectable change similar to CY857 because the GHR fusion protein islimiting. A compound interacting with the ligand fusion demonstrates theinverse change in measured phenotype in this secondary assay.

EXAMPLE 3 Demonstration of Ligand Dependent Receptor Dimerization

Multiple protein interactions (for example; ligand-receptor-receptor)are investigated with the expanded system which expresses a thirdprotein using the following scheme.

One unit of the receptor dimer is generated as a fusion protein witheither the Gal 4 DNA binding or activation domain. The other unit of thereceptor dimer is generated as a fusion protein with corresponding GalDNA binding or activation domain, whichever is not used for the firstfusion. The gene encoding the ligand is expressed from the third plasmidand is produced as a free (non-fusion) ligand. Interaction of the fusionproteins occurs only in the presence of ligand (see FIG. 3).

The interaction of vascular endothelial cell growth factor (VEGF) withthe ligand binding domain of its cognate receptor (KDR, kinase insertdomain containing receptor) is described as an example for this system.KDR is a tyrosine kinase receptor, and dimer formation (1 ligand-2receptors) is suggested to be important for hormone-induced receptorfunction. The cDNA encoding the ligand domain of KDR (Terman et al.,1991) is isolated as an Nco I-BamHI fragment and cloned into both thepACT-II and pAS2 vectors. The cDNA encoding the mature protein for VEGFis generated using standard PCR techniques. Oligonucleotides aredesigned from published sequence (see Tischer et al., 1991). A 34 base5′ oligonucleotide containing an EcoRI site(5′-CGGAATTCGAAGTATGGCACCCATGGCAGAAGGA-3′ SEQ ID NO: 14) and a 28 base3′ oligonucleotide containing an EcoRI site(5′-CGGAATTCGGATCCTCATTCATTCATCA-3′ SEQ ID NO: 15) are used to generatea 450 bp fragment encoding the mature protein and cloned into the EcoRIsite of pCUP. DNA of final recombinant vectors is transformed into yeastby the lithium acetate method to generate appropriate strains.

The yeast host strain (CY770) is transformed with KR-pACT-II, KDR-pAS2and VEGF-pCUP to generate strain CY846; or transformed with bothreceptor fusions and pCUP to generate strain CY847. Additionally, bothKR-pACT-II and KDR-pAS2 are transformed together (CY845) or separately(CY843 or CY844) or VEGF-pCUP alone (CY841) as control strains. Strainsare tested for growth on selective medium. The strain (CY846) thatexpresses the VEGF ligand plus the two receptor fusion proteins exhibitssubstantial growth on selective media in comparison to the strain CY847,which does not express the VEGF ligand (see FIG. 4). These resultsdemonstrate that the effective cells of this invention can be used tostudy ligand-dependent dimerization of the receptor.

EXAMPLE 4 Screen for Compounds That act as Ligands in a Dimer ReceptorSystem

Dimerization (oligomerization) of receptor units is often an importantfirst step in activation of receptors such as those for the growthfactors, cytokines and those describe supra. The novel cell systemdescribed in example 3 can be applied to the discovery of novelcompounds which promote (or block) receptor dimerization. Such novelinteracting compounds may serve as effective therapeutic agents forpathologies associated with these receptors.

Plasmids expressing the dimer receptor unit(s) as fusion proteins aregenerated as discussed in example 3. The strain (CY845) containing theKDR-pACT-II and KR-pAS2 fusions serves as an example of a simple primaryscreen for receptors which exhibit a dimer structure. Strain CY845 isembedded in synthetic agar medium deficient in histidine (Rose et al.,1990). Test compounds are applied to the top of this test medium.Chemical compounds which induce interaction of the two receptor fusions(in the absence of ligand) results in the reconstitution of theendogenous transcriptional activator, which is linked to a reportergene, such as HIS3. The reconstitution is identified by growth of cellssurrounding the compound.

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Li, J J and I. Herskowitz (1993) Isolation of ORC6, a component of theyeast origin recognition complex by a one-hybrid system. Science 262:1870-1874.

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What we claim is:
 1. A method of detecting the ability of a test sampleto affect the binding interaction of a first peptide and a secondpeptide of a peptide binding pair that bind through extracellularinteraction in their natural environment, comprising: (i) culturing atleast one cell, wherein the cell comprises; a) a nucleotide sequenceencoding a first heterologous fusion protein comprising the firstpeptide or a segment thereof joined to a transcriptional activationprotein DNA binding domain; b) a nucleotide sequence encoding a secondheterologous fusion protein comprising the second peptide or a segmentthereof joined to a transcription activation protein transcriptionalactivation domain; wherein binding of the first peptide or segmentthereof and the second peptide or segment thereof reconstitutes atranscriptional activation protein; and c) a reporter gene activatedunder positive transcriptional control of the reconstitutedtranscriptional activation protein, wherein expression of the reportergene produces a selected phenotype; (ii) incubating a test sample withthe cell under conditions suitable to detect the selected phenotype; and(iii) detecting the ability of the test sample to affect the bindinginteraction of the peptide binding pair by determining whether the testsample affects the expression of the reporter gene which produces theselected phenotype.
 2. The method of claim 1 wherein the cell furthercomprises at least one endogenous nucleotide sequence selected from thegroup consisting of a nucleotide sequence encoding the transcriptionalactivation protein DNA binding domain, a nucleotide sequence encodingthe transcriptional activation protein transcriptional activationdomain, and a nucleotide sequence encoding the reporter gene, wherein atleast one of the endogenous nucleotide sequences is inactivated bymutation or deletion.
 3. The method of claim 1 wherein the peptidebinding pair comprises a ligand and a receptor to which the ligandbinds.
 4. The method of claim 1 wherein the transcriptional activationprotein is Gal4, Gcn4, Hap2, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1,Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.
 5. The methodof claim 1 wherein the reporter gene is selected from the groupconsisting of lacZ, a gene encoding luciferase, a gene encoding greenfluorescent protein, and a gene encoding chloramphenicolacetyltransferase.
 6. The method of claim 1 wherein the peptide bindingpair is other than an antigen and a corresponding antibody.
 7. Themethod of claim 1 wherein the cell is a eukaryotic, vertebrate,mammalian, amphibian, Xenopus, fungal, Aspergillus, Neurospora, yeast,Schizosaccharomyces pombe, or Pichia pastoris cell.
 8. The method ofclaim 1 wherein the DNA binding domain is heterologous transcriptionalactivation protein DNA-binding domain.
 9. The method of claim 8 whereinthe DNA binding protein is selected from the group consisting of amammalian steroid receptor and bacterial LexA protein.
 10. The method ofclaim 1 wherein at least one of the heterologous fusion proteins isexpressed from an autonomously-replicating plasmid.
 11. The method ofclaim 10 wherein at least one peptide of the peptide binding pair isselected from the group consisting of a cytokine, an interleukin, ahematopoietic growth factor, insulin, an insulin-like growth factor, agrowth hormone, prolactin, an interferon, a growth factor, a ligand forG-protein coupled receptors, a ligand for guanylyl cyclase receptors, aligand for tyrosine phosphatase receptors, and a ligand for tyrosinekinase receptors.
 12. The method of claim 11 wherein the peptide is agrowth factor selected from the group consisting of epidermal growthfactor, nerve growth factor, leukemia inhibitory factor, fibroblastgrowth factor, platelet-derived growth factor, vascular endothelialgrowth factor, tumor necrosis factor, oncostatin M, ciliary neurotrophicfactor, erythropoietin, steel factor, placental lactogen, and TGF.
 13. Arescue screen for detecting the ability of a test sample to affect thebinding interaction of a first peptide and a second peptide of a peptidebinding pair, comprising: (i) culturing at least one cell, wherein thecell comprises; a) a nucleotide sequence encoding a first heterologousfusion protein comprising the first peptide or a segment thereof joinedto a transcriptional activation protein DNA binding domain; b) anucleotide sequence encoding a second heterologous fusion proteincomprising the second peptide or a segment thereof jointed to atranscriptional activation protein transcriptional activation domain;wherein binding of the first peptide or segment thereof and the secondpeptide or segment thereof reconstitutes a transcriptional activationprotein; and c) a reporter gene activated under positive transcriptionalcontrol of the reconstituted transcriptional activation protein, whereinexpression of the reporter gene prevents exhibition of a selectedphenotype; (ii) incubating a test sample with the cell under conditionssuitable to detect the selected phenotype; and (iii) detecting theability of the test sample to affect the binding interaction of thepeptide binding pair by determining whether the test sample affects theexpression of the reporter gene which prevents exhibition of theselected phenotype.
 14. The method of claim 13 wherein the cell furthercomprises at least one endogenous nucleotide sequence selected from thegroup consisting of a nucleotide sequence encoding the transcriptionalactivation protein DNA binding domain, a nucleotide sequence encodingthe transcriptional activation protein transcriptional activationdomain, and a nucleotide sequence encoding the reporter gene, wherein atleast one of the endogenous nucleotide sequences is inactivated bymutation or deletion.
 15. The method of claim 13 wherein the peptidebinding pair comprises a ligand and a receptor for the ligand.
 16. Themethod of claim 13 wherein the transcriptional activation protein isGal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Prp1, Arg81,Lac9, Qa1F, VP16, or a mammalian nuclear receptor.
 17. The method ofclaim 13 wherein the receptor gene is selected from the group consistingof a gene that prevents growth on cycloheximide and a gene that preventsgrowth on canavanine.
 18. The method of claim 13 wherein the peptidebinding pair is other than an antigen and a corresponding antibody. 19.The method of claim 13 wherein the cell is a eukaryotic, vertebrate,mammalian, amphibian, Xenopus, fungal, Aspergillus, Neurospora, yeast,Schizosaccharomyces pombe, or Pichia pastoris cell.
 20. The method ofclaim 13 wherein the DNA binding domain is a heterologoustranscriptional activation protein DNA-binding domain.
 21. The method ofclaim 20 wherein the DNA binding protein is selected from the groupconsisting of a mammalian steroid receptor and bacterial LexA protein.22. The method of claim 13 wherein at least one of the heterologousfusion proteins is expressed from an autonomously-replicating plasmid.23. The method of claim 22 wherein at least one peptide of the peptidebinding pair is selected from the group consisting of a cytokine, aninterleukin, a hematopoietic growth factor, insulin, an insulin-likegrowth factor, a growth hormone, prolactin, an interferon, a growthfactor, a ligand for G-protein coupled receptors, a ligand for guanylylcyclase receptors, a ligand for tyrosine phosphatase receptors, and aligand for tyrosine kinase receptors.
 24. The method of claim 23 whereinthe peptide is a growth factor selected from the group consisting ofepidermal growth factor, nerve growth factor, leukemia inhibitoryfactor, fibroblast growth factor, platelet-derived growth factor,vascular endothelial growth factor, tumor necrosis factor, oncostatin M,ciliary neurotrophic factor, erythropoietin, steel factor, placentallactogen, and TGF.
 25. A rescue screen for determining whether a testsample interacts with a first or second peptide of a peptide bindingpair, comprising: (i) culturing at least one first cell, wherein thefirst cell comprises; a) a nucleotide sequence encoding a firstheterologous fusion protein comprising the first peptide or a segmentthereof joined to a transcriptional activation protein DNA bindingdomain; b) a nucleotide sequence encoding a second heterologous fusionprotein comprising the second peptide or a segment thereof joined to atranscriptional activation protein transcriptional activation domain;wherein the nucleotide sequence encoding the first heterologous fusionprotein is present in an effective copy number of at least 5 copies percell and the nucleotide sequence encoding the second heterologous fusionprotein is present at a copy number of 1 or 2 per cell; and whereinbinding of the first peptide or segment thereof and the second peptideor segment thereof reconstitutes a transcriptional activation protein;and c) a reporter gene activated under positive transcriptional controlof the reconstituted transcriptional activation protein, whereinexpression of the reporter gene prevents exhibition of a selectedphenotype; (ii) culturing at least one second cell, wherein the secondcell comprises; a) a nucleotide sequence encoding a first heterologousfusion protein comprising the first peptide or a segment thereof joinedto a transcriptional activation protein DNA binding domain; b) anucleotide sequence encoding a second heterologous fusion proteincomprising the second peptide or a segment thereof joined to atranscriptional activation protein transcriptional activation domain;wherein the nucleotide sequence encoding the second heterologous fusionprotein is present in an effective copy number of at least 5 copies percell and the nucleotide sequence encoding the first heterologous fusionprotein is present at a copy number of 1 or 2 per cell; and whereinbinding of the first peptide or segment thereof and the second peptideor segment thereof reconstitutes a transcriptional activation protein;and c) a reporter gene activated under positive transcriptional controlof the reconstituted transcriptional activation protein, whereinexpression of the reporter gene prevents exhibition of a selectedphenotype; (iii) incubating a test sample with the first and secondcells under conditions suitable to detect the selected phenotype; (iv)detecting the presence or absence of the selected phenotype produced bythe first and second cells; and (v) comparing the selected phenotype ofthe first and second cells, wherein a change in the selected phenotypein one of the cells indicates that the test sample binds to theheterologous fusion protein encoded by the nucleotide sequence presentat a copy number of 1 or 2 in that cell exhibiting the change in theselected phenotype, thereby affecting the binding interaction of thepeptide binding pair.
 26. The method of claim 25 wherein the cellfurther comprises at least one endogenous nucleotide sequence selectedfrom the group consisting of a nucleotide sequence encoding thetranscriptional activation protein DNA binding domain, a nucleotidesequence encoding the transcriptional activation protein transcriptionalactivation domain, and a nucleotide sequence encoding the reporter gene,wherein at least one of the endogenous nucleotide sequences isinactivated by reconstitution or deletion.
 27. The method of claim 25wherein the peptide binding pair comprises a ligand and a receptor forthe ligand.
 28. The method of claim 25 wherein the transcriptionalactivation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1 ,Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.29. The method of claim 25 wherein the DNA binding protein is selectedfrom the group consisting of a gene that prevents growth oncycloheximide and a gene that prevents growth on canavanine.
 30. Themethod of claim 25 wherein the peptide binding pair is other than anantigen and a corresponding antibody.
 31. The method of claim 25 whereinthe first cell and the second cell are the same are a eukaryotic,vertebrate, mammalian, amphibian, Xenopus, fungal, Aspergillus,Neurospora, yeast, Schizosaccharomyces pombe, or Pichia pastoris cell.32. The method of claim 25 wherein the DNA binding domain is aheterologous transcriptional activation protein DNA-binding domain. 33.The method of claim 32 wherein the DNA binding protein is selected fromthe group consisting of a mammalian steroid receptor and bacterial LexAprotein.
 34. The method of claim 25 wherein at least one of theheterologous fusion proteins is expressed from anautonomously-replicating plasmid.
 35. The method of claim 34 wherein atleast one peptide of the peptide binding pair is selected from the groupconsisting of a cytokine, an interleukin, a hematopoietic growth factor,insulin, an insulin-like growth factor, a growth hormone, prolactin, aninterferon, a growth factor, a ligand for G-protein coupled receptors, aligand for guanylyl cyclase receptors, a ligand for tyrosine phosphatasereceptors, and a ligand for tyrosine kinase receptors.
 36. The method ofclaim 35 wherein the peptide is a growth factor selected from the groupconsisting of epidermal growth factor, nerve growth factor, leukemiainhibitory factor, fibroblast growth factor, platelet-derived growthfactor, vascular endothelial growth factor, tumor necrosis factor,oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor,placental lactogen, and TGF.
 37. A rescue screen for detecting theability of a test sample to affect the binding interaction of a firstand second peptide of a peptide binding pair, comprising: (i) culturingat least one cell on a selective medium, wherein the cell comprises; a)a nucleotide sequence encoding a first heterologous fusion proteincomprising the first peptide or a segment thereof joined to atranscriptional activation protein DNA binding domain; b) a nucleotidesequence encoding a second heterologous fusion protein comprising thesecond peptide or a segment thereof joined to a transcriptionalactivation protein transcriptional activation domain; wherein binding ofthe first peptide or segment thereof and the second peptide or segmentthereof reconstitutes a transcriptional activation protein; c) areporter gene activated under positive transcriptional control of thereconstituted transcriptional activation protein, wherein when thereporter gene is expressed the cell does not grow on the selectivemedium; and (ii) incubating a test sample with the cell; and (iii)detecting the ability of the test sample to affect the bindinginteraction of the peptide binding pair by determining whether the testsample affects the expression of the reporter gene which prevents growthof the cell on the selective medium.
 38. The method of claim 37 whereinthe cell further comprises at least one endogenous nucleotide sequenceselected from the group consisting of a nucleotide sequence encoding thetranscriptional activation protein DNA binding domain, a nucleotidesequence encoding the transcriptional activation protein transcriptionalactivation domain, and a nucleotide sequence encoding the reporter gene,wherein at least one of the endogenous nucleotide sequences isinactivated by mutation or deletion.
 39. The method of claim 37 whereinthe peptide binding pair comprises a ligand and a receptor for theligand.
 40. The method of claim 37 wherein the transcriptionalactivation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1,Ace1, Prp1, Arg81, Lac9, Qa1F, VP16, or a mammalian nuclear receptor.41. The method of claim 37 wherein the reporter gene is selected fromthe group consisting of a gene that prevents growth on cycloheximide anda gene that prevents growth on canavanine.
 42. The method of claim 37wherein the peptide binding pair is other than an antigen and acorresponding antibody.
 43. The method of claim 37 wherein the firstcell and the second cell are the same and are a eukaryotic, vertebrate,mammalian, amphibian, Xenopus, fungal, Aspergillus, Neurospora, yeast,Schizosaccharomyces pombe, or Pichia pastoris cell.
 44. The method ofclaim 37 wherein the DNA binding domain is a heterologoustranscriptional activation protein DNA-binding domain.
 45. The method ofclaim 44 wherein the DNA binding protein is selected from the groupconsisting of a mammalian steroid receptor and bacterial LexA protein.46. The method of claim 37 wherein at least one of the heterologousfusion proteins is expressed from an autonomously-replicating plasmid.47. The method of claim 46 wherein at least one peptide of the peptidebinding pair is selected from the group consisting of a cytokine, aninterleukin, a hematopoietic growth factor, insulin, an insulin-likegrowth factor, a growth hormone, prolactin, an interferon, a growthfactor, a ligand for G-protein coupled receptors, a ligand for guanylylcyclase receptors, a ligand for tyrosine phosphatase receptors, and aligand for tyrosine kinase receptors.
 48. The method of claim 47 whereinthe peptide is a growth factor selected from the group consisting ofepidermal growth factor, nerve growth factor, leukemia inhibitoryfactor, fibroblast growth factor, platelet-derived growth factor,vascular endothelial growth factor, tumor necrosis factor, oncostatin M,ciliary neurotrophic factor, erythropoietin, steel factor, placentallactogen, and TGF.
 49. A rescue screen for determining whether a testsample interacts with a first or second peptide of a peptide bindingpair, comprising: (i) culturing at least one first cell, wherein thefirst cell comprises; a) a nucleotide sequence encoding a firstheterologous fusion protein comprising the first peptide or a segmentthereof joined to a transcriptional activation protein DNA bindingdomain; b) a nucleotide sequence encoding a second heterologous fusionprotein comprising the second peptide or a segment thereof joined to atranscriptional activation protein transcriptional activation domain;wherein the nucleotide sequence encoding the first heterologous fusionprotein is present in an effective copy number of at least 5 copies percell and the nucleotide sequence encoding the second heterologous fusionprotein is present at a copy number of 1 or 2 per cell; and whereinbinding of the first peptide or segment thereof and the second peptideor segment thereof reconstitutes a transcriptional activation protein;and c) a reporter gene activated under positive transcriptional controlof the reconstituted transcriptional activation protein, wherein whenthe reporter gene is expressed the cell does not grow on the selectivemedium; (ii) culturing at least one second cell, wherein the second cellcomprises; a) a nucleotide sequence encoding a first heterologous fusionprotein comprising the first peptide or a segment thereof joined to atranscriptional activation protein DNA binding domain; b) a nucleotidesequence encoding a second heterologous fusion protein comprising thesecond peptide or a segment thereof joined to a transcriptionalactivation protein transcriptional activation domain; wherein thenucleotide sequence encoding the second heterologous fusion protein ispresent in an effective copy number of at least 5 copies per cell andthe nucleotide sequence encoding the first heterologous fusion proteinis present at a copy number of 1 or 2 per cell; and wherein binding ofthe first peptide or segment thereof and the second peptide or segmentthereof reconstitutes a transcriptional activation protein; and c) areporter gene activated under positive transcriptional control of thereconstituted transcriptional activation protein, wherein when thereporter gene is expressed the cell does not grow on the selectivemedium; (iii) incubating a test sample with the first and second cellsunder conditions suitable to detect the selected phenotype; (iv)detecting the presence or absence of cell growth by the first and secondcells; and (v) comparing the growth of the first and second cells,wherein growth of one of the cells indicates that the test sample bindsto the heterogeneous fusion protein encoded by the nucleotide sequencepresent at a copy number of 1 or 2 in the growing cell, therebyaffecting the binding interaction of the peptide binding pair.
 50. Themethod of claim 49 wherein the cell further comprises at least oneendogenous nucleotide sequence selected from the group consisting of anucleotide sequence encoding the transcriptional activation protein DNAbinding domain, a nucleotide sequence encoding the transcriptionalactivation protein transcriptional activation domain, and a nucleotidesequence encoding the reporter gene, wherein at least one of theendogenous nucleotide sequences is inactivated by mutation or deletion.51. The method of claim 49 wherein the peptide binding pair comprises aligand and a receptor for the ligand.
 52. The method of claim 49 whereinthe transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5,Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammaliannuclear receptor.
 53. The method of claim 48 wherein the reporter geneis selected from the group consisting of a gene that prevents growth oncycloheximide and a gene that prevents growth on canavanine.
 54. Themethod of claim 49 wherein the peptide binding pair is other than anantigen and a corresponding antibody.
 55. The method of claim 49 whereinthe first cell and the second cell are the same and are a eukaryotic,vertebrate, mammalian, amphibian, Xenopus, fungal, Aspergillus,Neurospora, yeast, Schizosaccharomyces pombe, or Pichia pastoris cell.56. The method of claim 49 wherein the DNA binding domain is aheterologous transcriptional activation protein DNA-binding domain. 57.The method of claim 56 wherein the DNA binding protein is selected fromthe group consisting of a mammalian steroid receptor and bacterial LexAprotein.
 58. The method of claim 49 wherein at least one of theheterologous fusion proteins is expressed from anautonomously-replicating plasmid.
 59. The method of claim 58 wherein atleast one peptide of the peptide biding pair is selected from the groupconsisting of a cytokine, an interleukin, an hematopoietic growthfactor, insulin, an insulin-like growth factor, a growth hormone,prolactin, an interferon, a growth factor, a ligand for G-proteincoupled receptors, a ligand for guanylyl cyclase receptors, a ligand fortyrosine phosphatase receptors, and a ligand for tyrosine kinasereceptors.
 60. The method of claim 59 wherein the peptide is a growthfactor selected from the group consisting of epidermal growth factor,nerve growth factor, leukemia inhibitory factor, fibroblast growthfactor, platelet-derived growth factor, vascular endothelial growthfactor, tumor necrosis factor, oncostatin M, ciliary neurotrophicfactor, erythropoietin, steel factor, placental lactogen, and TGF.